Figure 1. Western blot analysis of Melanoma gp100 using anti-Melanoma gp100 antibody.
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel)/90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: rat heart tissue lysate,
Lane 2: mouse heart tissue lysate.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Melanoma gp100 antigen affinity purified polyclonal antibody at 0.5 ug/ml overnight at 4 degree C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for Melanoma gp100 at approximately 70KD. The expected band size for Melanoma gp100 is at 70KD.